One of the most common methods for nucleic acid
detection and quantification is the measurement of solution absorbance at 260
nm (A260) due to the fact that nucleic acids have an absorption maximum at this
UV wavelength
When DNA is present in the sample a fraction of the ultraviolet light will pass through
and an other fraction will be absorbed and the amount of the light absorbed is directly
proportional to the nucleic acid concentration in the sample. Using the Beer-Lambert Law it
is possible to relate the amount of light absorbed to the concentration of the
absorbing molecule.
At a wavelength of 260nm, the average extinction coefficient is:
-
0.020
(μg/ml)−1cm−1, double-stranded DNA;
-
0.027
(μg/ml)−1 cm−1, for single-stranded DNA
Spectrophotometric
quantification is precise and with the advent of microvolume spectrophotometer (e.g nanodrop) those allow to perform measurement using
very small sample volumes (1-2ul) with-out the use of any sample support (e.g quartz
cuvette) it become the method of first choice
for DNA plasmid quantification in the molecular biology laboratories.
DNA quantification with microvolume spectrophotometer is precise and allow to evaluate DNA purity and RNA but it is time consuming (20-30’’ for sample) and therefore not applicable to the measurement of a huge number of samples in parallel.
Modern HT (High throughput) cloning platforms produce hundreds of DNA samples (plasmidic ans/or genomic) in parallel and using a multiwell based approach is certainly preferable to speed up the process.
Since multiwell determination require at least 50-100 ul of sample/well, a preliminary sample dilution step is required to do not use the entire DNA sample for this step, but this may represent a problem, since the method sensitivity is limited.
For example:
- If we consider that A(260)=0.1 using a spectrophotometer with 1 cm of optical path-lenght correspond to a dna sample with concentration of 5ng/ul.
Generally the path-length in a multiwell plates is lower than 1cm
For example 100ul of an half area UVclear 96 well plate result on a path length of about 0,67cm (A(260)=0,1 with a 7,5ng/ul sample)
Therefore if we dilute 10ul of our MINI PREP to 100ul final and we read the ABS280 on a multiplate reader we will obtain a detectable ABS (>0,1) only for samples with concentration >75ng/ul that is too high since in my experience the range of plasmid concentrations that are generally obtained with a 96 well plate mini kit is in the range 20-100ng/ul.
In this post I would like to show you as using a common Fluorimetric stains (in my case Lonza Gel Star) developed to bind DNA staining in agarose gel a rapid DNA quantification could be performed in 96 well plate format.
Example:
DNA plasmidic quantification using Gel
Star probe (Lonza)
2ul DNA sample in 100ul
Gelstar stain diluted 10000 times (1X final concentration) in H2O
plates: 96 well flat black
(Greiner)
Instrument: multiplate reader (Tecan M200) Ex:490nm; Em:530mn (gain:80)
A standard
curve was built using an available commercial plasmid pRSET/BFP (Invitrogen)
and serial dilution were performed to obtain a final DNA concentration range
(2,5- 0,0025ug/ml)
The fluorimetric methods using GelStar show linearity in a concentration range 0,01ng/ul to 0,625ng/ul.
Therefore the methods, using 2ul of dna sample (dilute in 100 of probe) could be direclty applied to the quantification of DNA samples in concentration range 0,5-30 ng/ul that is in the range of the sample that normally are obtained for 96-well mini kit dna preparation kit.
In case that DNA samples are more concentrated we can simple reduce the DNA volumes (to 1ul or 0,5ul) used for the test.
Considering that fluorescence of the probe can depend from DNA size and origin (single or double strand) is it ever suggested to perform a calibration line with a standard DNA sample with similar size and origin respect the ones that we would like to quantify.
Of course differently to 260/280 nm quantification this methods do not allow to you to estimate sample purifity )in terms of protiens) or buffer contamination but i'm my opinion modern mini kits are quite reilable and in 99% of cases the sample quality is ok for the downstream applications (eg sequencing, E.coli trasformation)
In my experience, this method is very usefull to rapid quantification of high number of purified plasmid to use for sequencing, trasformation and protein expression.
I have done those trials with GelStar probe since it was the one avaialble in my lab at the time of this test but i suppose that similar results can be obtained also with other simiilar probes (eg, Gelred, midori green, Sybr safe it the right Ex/Em wavelenght were selected. Since each probe is chatacterized from a different fluoresence quantum yielad is possible that a different probe may affect a little the limit of sensitivity.
Please, DO NOT USE Ethidium Bromide!!
Fortunatelly today, less toxic probes (as i already mentioned in the
ProteoCool Pills n°4) with similar sentitivity and low cost are avaialble.
